Signaling compositions, methods, and systems for effecting plant burndown and herbicide enhancement

ABSTRACT

Bio-regulators from a group of quaternary ammonium moieties modify a gene expression in undesirable plants to inhibit growth and robustness and enhance the effectiveness of herbicides. Such bio-regulator may be applied to plants through seed treatments, root drenching, spraying and dusting, or to soil were desirable crops are planted or will be planted. Bio-regulators may be duel-acting; causing beneficial modification to gene expressions in desirable plants while modifying gene expression in undesirable plants, making them more susceptible to herbicides. Bio-regulators are Ester Compounds, BMIA Compounds or related salts of those compounds.

FIELD OF INVENTION

The present invention relates to bio-regulation in plants, and moreparticularly to compositions for inducing valuable qualities in plantssuch as earlier germination, increased vigor, earlier or delayedmaturity, improved crop yield, tolerance to biotic and abiotic cropproduction stresses and enhancement of herbicidal activity.

BACKGROUND

Global food production has increased steadily and yet there are stillcountries with malnourished populations. Many of these countries rely onagriculture, and have not seen an increase in overall food production inspite of global increases. Some of these countries cannot grow thevarious foods required to meet nutrition requirements due to human andenvironmental forces.

With advancements in technology, people live longer, birth rates arehigher, and population increases place additional stress on resources.As the population continues to increase, so will the demand for food,fiber, and agriculture based renewable energy sources. With an increasedpopulation comes the requirement for additional housing andinfrastructure. This increase decreases the amount of acreage availablefor effective crop production. Therefore, there is an increasing demandfor agricultural crop products, while the available land to produce suchproducts is limited. Other concerns include depletion of nutrients fromthe available soil, the effectiveness of current agricultural productionmethods, environmental impacts on plants, and loss of arable landresulting from increased global warming (which has yet to be adequatelystudied).

With these growing concerns, it is necessary for society to continuallydevelop advancements in sustainably increasing production from limitedarable resources. It is also evident that cost effective methods forincreased agricultural production and crop yield must undergo continueddevelopment.

Attempts to address some of these problems have included methods such asbreeding and selecting more productive plant varieties; improved cropmanagement; advances in technology; use of fertilizers, herbicides, andpesticides; and changes in irrigation techniques. These methods havebeen useful in some countries, but have had limited impact on developingnations where cultural practices and farm management are not advancedand where the cost of fertilizer or lack of irrigation practices haveproduced less than satisfactory crop yields. Even in countries withadvanced farm management techniques, such as the United States,continual advances in productivity are necessary, and such necessity maynot be satisfied by known compositions and methods.

Genetic engineering techniques are being employed to produce plants thatare tolerant or resistant to environmental stresses and pathogen andpest pressure (abiotic and biotic stresses, respectively). Geneticengineering of plants for crop production often involves identificationand insertion of foreign genes. The practice is time consuming, costlyand often limiting with regards to numbers of genes that can be insertedor activated in a genetically engineered plant.

Insufficient time and attention has been devoted to the control ofstress in biological processes at the cellular level. Stress is relevantto crop productivity and quality. Plants have the biological potentialto deal with environmental stress in a fashion which may be exploitedfor the sustainable benefit of humankind. Compositions and methods forusing such compositions to modify gene expression in plants to improvecrop yields under various environmental stresses are needed.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a novelmethod for manipulating the levels of gene products that are involved inpersistence of useful and elimination of non-useful plants in thepresence and absence of abiotic and biotic stresses.

It is another object of the present invention to provide the proceduresnecessary for performing the manipulations.

It is still another object of the present invention to provide thenecessary components for inducing the manipulated stress levels.

It is still another object of the present invention to developinexpensive methods and procedures that can be produced on large andsmall scales.

It is still another object of the present invention to provideappropriate compositions and families of compositions to be incorporatedinto the methods and procedures.

It is still another object of the present invention to provideappropriate wetting agents and penetrating adjuvants to be used in themethods and procedures.

It is still another object of the present invention to providebio-regulator solutions formed from mixing amounts of said compositionsand appropriate wetting agents and penetrating adjuvants to be used inthe methods and procedures.

It is still another object of the present invention to provide variousmethods of applying said compositions to sexual and vegetative parts ofplants and to microorganisms.

In accordance with the above objects of the invention, in one embodimentof the present invention, several families of compositions are appliedaccording to the methods and procedures outlined. The compositions areapplied in bio-regulatory active concentrations to seeds and/or plants,sufficient to utilize, enhance or inhibit internal and external plantcharacteristics, even while the plant may be subjected to various stressconditions such as high insect and microbial pests, saline environments,anaerobic environments, high temperatures, low temperatures and droughtconditions. Some of the internal characteristics utilized, enhanced orinhibited include, but are not limited to, abiotic and biotic stressresponse gene products, sugar, essential oil, Vitamin C, carotenoids andprotein content. Some external characteristics utilized, enhanced orinhibited include, but are not limited to, vigor, pigment accumulation,rate of development, and yield.

In another embodiment, the compositions keep the plants in abio-regulated state sufficient to increase or enhance the treated plantsability to grow. The applied compositions are selected from the group ofchemical compounds represented by the formula:

wherein R1 and R2 are preferably lower alkyl groups containing one tosix carbon atoms, inclusive, or a benzyl substituent each of identicalor dissimilar structure; n is an integer (preferably from zero to four);R3 is selected from the group comprising lower alkyl compositionscontaining from one to six carbon atoms (preferably includingcyclopropyl, phenyl, and alkyl substituted phenyl). This family ofcompositions is referred to herein as the “Ester Compounds” and may alsoinclude acid addition salts of such compounds.

In another embodiment, the applied compositions are selected from thegroup represented by the formula:

or a substantially similar molecules. This family of compounds, as morefully defined herein, is referred to herein as the “BMIA Compounds”.

In another embodiment, the applied compositions are also selected fromthe group of salts resulting from the Ester Compounds and BMIA Compounds(preferably salts resulting from halides—i.e. chloro and bromo salts).This family of compositions is referred to herein as the “SaltCompounds”.

In another embodiment the applied compositions are selected from acombination of the Ester Compounds and their Salt Compounds, or from acombination of the BMIA Compounds and their Salt Compounds.

In another embodiment the applied compositions are selected from thepreviously disclosed groups in bio-regulatory concentrations incombination with herbicide compositions sufficient to decrease orinhibit a treated plant's ability to grow, or to increase the herbicidalactivity. In this embodiment the method is preferably used for killingundesirable plants and weeds, such as during burndown.

In another embodiment, amounts of these compositions are applied inbio-regulatory concentrations to form bio-regulator solutions, which areapplied according to various means. In another embodiment, the range ofconcentrations of compositions used to generate the bio-regulatorsolutions is between 0.05 and 2000 parts per million (“ppm”). Seedtreatment solutions may be used in a very low dose total volume, buthigh A.I. concentration, applications.

In another embodiment, the present invention includes, soaking,spraying, dusting and coating plants and seeds. These applicationmethods may be utilized for both the sexual reproductive and vegetativeparts of plants.

In another embodiment, seed treatment includes incorporating the plantbio-regulators into seed priming systems.

In a seed priming system, seeds are partially hydrated and maintainedunder defined moisture, temperature and aeration conditions for aprescribed period of time. During priming, seed metabolic activityfacilitates completion of important pre-germination steps like membranerepair, DNA and RNA synthesis and repair, expression of particular geneproducts that are involved in seed germination and derived plantdevelopment over a range of environments, development of immatureembryos, alteration of tissues covering the embryo, and removal ofdormancy blocks to advance germination status. After priming, seeds aregenerally dried back to storage moisture contents so they can be plantedwith conventional equipment at a later date.

Seeds treated in a priming system have fewer steps to complete thannon-primed seeds in order to accomplish germination. Consequently,priming improves the rate and uniformity of seed germination.Additionally, seed repair and/or removal of dormancy blocks duringpriming of a particular seed lot can result in increased germinationpercentage. The improvements priming offers to seed germinationcharacteristics often translate to more rapid and uniform seedlingemergence, increased uniformity and rates of plant development, andincreased yields.

In this embodiment the method is preferably used to enhance or inhibitseed metabolic activity that is made amenable to bio-regulator compoundactivity as a function of the metabolic status achieved and/ormaintained during and/or following the seed priming process to influenceseed germination and derived plant growth and development under a rangeof environments that include the presence and absence of abiotic and/orbiotic stress.

In another embodiment, seed treatment includes incorporating the plantbio-regulators into seed coating systems. Seed coating involvesapplication of certain active ingredients, including but not limited tofungicides, insecticides, plant growth promoting compounds, beneficialmicrobes, to seeds with a binder and other inert materials to facilitatemechanical planting of seeds and protect the seeds once they areplanted.

In this embodiment the method is preferably used to enhance or inhibitseed metabolic activity to influence seed germination and derived plantgrowth and development under a range of environments that include thepresence and absence of abiotic and/or biotic stress. Further, in thisembodiment, the method of incorporating the plant bio-regulators intoseed coating systems is preferably used to augment incorporation of theplant bio-regulators into seed priming systems.

In another embodiment a method of seed treatment is encapsulation of theseed with a material forming the capsule surrounding the seed. Examplesof encapsulation systems include interfacial polymerization, in-situpolymerization, matrix polymerization and the like. For example, inmatrix polymerization a core material is imbedded in a polymeric matrixduring formation of the particles. A simple method of this type isspray-drying, in which the particle is formed by evaporation of thesolvent from the matrix material. However, the solidification of thematrix also can be caused by a chemical change. In Interfacialpolymerization, the two reactants in a polycondensation meet at aninterface and react rapidly. In yet another embodiment, in situpolymerization the direct polymerization of a single monomer is carriedout on the particle surface.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not necessarilyrestrictive of the invention as claimed. The accompanying drawings,which are incorporated in and constitute a part of the specification,illustrate embodiments of the invention and with the generaldescription, explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows a general chemical structure of a compound according toat least one embodiment of the present invention;

FIG. 1 b shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 c shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 d shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 e shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 f shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 g shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 h shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 i shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 j shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 k shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 l shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 m shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 n shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 1 o shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 2 a shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 2 b shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 2 c shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 2 d shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 2 e shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 2 f shows a chemical structure of a compound according to at leastone embodiment of the present invention;

FIG. 3 shows a flowchart for a method of testing concentrations of abio-regulator to modify a gene expression;

FIG. 4 shows a flowchart for a method of treating a crop to modify agene expression;

FIG. 5 shows a flowchart for a method of testing concentrations of abio-regulator to modify a gene expression based on a stressor;

FIG. 6 a shows nodes at which flowering occurred in plants treated withembodiments of the present invention;

FIG. 6 b shows nodes at which flowering occurred in plants treated withembodiments of the present invention;

FIG. 7 shows nodes at which pod formation occurred in plants treatedwith embodiments of the present invention; and

FIG. 8 shows yields from a number of the locations in units of bushelsof a study including embodiments of the present invention.

DETAILED DESCRIPTION

The present invention includes compositions, systems, and processes forenhancing or modifying expression of particular gene products to producedesirable improvement in specific plant properties related to internaland external plant characteristics and overall agricultural output. Themethods and processes are useful in seed encapsulation, coating, andsoaking; plant spraying and rhizosphere drenching. Further objects ofthe inventions will be evident from the following description, wherein,parts and percentages are by weight unless otherwise specified.

A benefit of the present invention can be obtained by applying thedescribed compositions in bio-regulatory amounts to the sexualreproductive parts and vegetative parts of plants, which can include butare not limited to seeds, bulbs, roots, seedlings, growing plants,slips, and cuttings (plants include trees). Practically, this can bedone via soaking plant parts in a solution containing the activebio-regulator composition; applying the bio-regulator solution duringthe seed priming process; applying the bio-regulator to seeds with abinder and other inert materials; dusting the seeds with a powdercontaining the bio-regulator composition; spraying the seed in furrowwith a bio-regulator solution or dusting the seed with a powderformulation containing the bio-regulator prior to covering the seed;encapsulation; spraying the cotyledon before transplanting or while inthe furrow; spraying the vegetative parts of the plant at various stagesof plant development; dusting the cotyledons before transplanting orwhile in the furrow; dusting various parts of the plant at variousstages of plant development; irrigating the roots or plants with solventmixed with bio-regulator compositions before transplanting or while inthe furrow; and through other methods of application.

Bio-regulator solutions are prepared by selecting compositions from thefollowing groups of classes of compositions:

A. Ester Compounds:

Referring to FIGS. 1 a-1 o, chemical compounds are shown that may beregarded as “Ester Compounds.” A person skilled in the art mayappreciate that Ester Compounds as represented by FIGS. 1 a-1 o mayencompass other compounds not specifically listed.

B. BMIA Compounds

Referring to FIGS. 2 a-2 f, chemical compounds are shown that may beregarded as “BMIA Compounds.” A person skilled in the art may appreciatethat BMIA Compounds as represented by FIGS. 2 a-2 f may encompass othercompounds not specifically listed.

Referring to FIG. 3, a flowchart for a method of testing concentrationsof a bio-regulator to modify expression of a gene is shown. Modifyinggene expression may influence plant phenotype in a growing locationgenerally subject to a certain range of temperatures, moisture,humidity, and chemical conditions; particularly, at a certain time ofyear, or growing season. The bio-regulatory effective amount of thecomposition to be applied may vary depending upon the mode ofapplication, stage of plant development and plant species. Determiningan appropriate concentration of bio-regulator may include selecting 300a seed or plant variety and selecting 302 a growing location. Seedvarieties and growing locations may generally have characteristics thatrequire specific formulations. Relevant characteristics of the growinglocations such as temperature variations, humidify and soil chemistrymay be determined 304. An appropriate application for the seed or plant,growing location and desired outcome may be determined 306. Abio-regulator is then selected 308 from the Ester Compounds, BMIACompounds and corresponding Salt Compounds described herein. A pluralityof testing samples is prepared 310. Each testing sample may replicate atleast some characteristics of the growing location. Where testing isconcerned with seed treating, the selected bio-regulator is applied 312via soaking, coating, priming or other seed treating methods. Wheretesting is concerned with developing plant treating, the selectedbio-regulator is applied 312 via spraying, dusting or other suchmethods. In either case, testing samples may be treated with theselected bio-regulator at varying concentrations to determine a minimumand maximum bio-regulatory effective concentration. After an appropriategrowth duration to assess the effectiveness of the bio-regulator, ananalysis is conducted 314 to determine if the selected bio-regulator waseffective and what concentrations or amounts produced the desiredresults.

Referring to FIG. 4, a flowchart for a method of treating a crop tomodify a gene expression is shown. Modifying one or more geneexpressions may generally enhance crop productivity. First, a crop isselected 400 or identified; then the growing location is selected 402 oridentified. Characteristics of the growing location such as temperature,moisture, humidity and soil chemistry are determined 404. Based on thosecharacteristics, a bio-regulator is selected 406 to enhance cropproductivity at the particular location. An application method isdetermined 408 based on the crop, growing location and desired result.Based on the determined application method, crop seeds are coated 410,crop plants are sprayed or dusted 412, or growing location soil istreated 414.

Referring to FIG. 5, a flowchart for a method of testing concentrationsof a bio-regulator to modify a gene expression based on a stressor isshown. Where a crop is undergoing or expected to undergo a specificstressor, a bio-regulator specific to the crop and the stressor may beused to enhance the robustness of the crop to the particular stressor.Specific stressors such as temperature variations, moisture, humidity orsoil chemistry are identified 500. A previously tested bio-regulator isselected 502 based on the stressor to modify one or more geneexpressions. An appropriate concentration of bio-regulator is determined504 based on previous testing and the concentration of bio-regulator isapplied 506 to the crop, either as seeds or growing plants.

In one exemplary test, microarray and illumina analyses were performedon monocot and dicot plant tissues treated with bio-regulator compoundsto see what genes were expressed as a result. Based on the types ofgenes expressed, it may be determined how plants treated with aparticular dose of a particular bio-regulator might perform in certainenvironmental conditions. For example bio-regulator compound treatmentson soybean, corn and Arabidopsis leaf tissue may result in expression ofgenes associated with heat, drought, and salt tolerance in plant tissue.

In another exemplary test, a large factorial screening test wasperformed where seeds and/or plants of a range of plant species weretreated with a dose range of each of a number of bio-regulatorsaccording to embodiments of the present invention and then grown out ina growth chamber set to a certain environmental stress condition or in apotting medium incorporating a stress component (i.e. NaCl). Phenotypeassessments (root and shoot dry weights, root-shoot ratios, plantheight, flowering characteristic ratings, etc. . . . ) may be doneduring and at the end of the grow-out. Such screening test may be thebasis for selecting a bio-regulator compound and dose that result inoptimum performance in that particular environment. Similar types ofscreening at a different grow-out scale may be performed to assessingperformance of plants treated with a bio-regulator and then exposed topathogenic microbe/insect pressure.

Application of the bio-regulator compounds to seeds prior to exposure ofthe seeds and resultant plants to abiotic and/or biotic stress and priorto resultant plants achieving growth stages in which yield factors suchas seed size and seed number are set, is preferred. Use of an activebio-regulator above certain threshold concentrations may inhibit growthof the resultant plants and even exhibit phytotoxic effects on theresultant plants, which in some circumstances may also be a desiredresult.

In at least one embodiment of the present invention, appropriate wettingagents and penetrating adjuvants are included in the composition. Suchagents may include but are not limited to: Triton X-100 (polyoxythyleneglycol p-isooctylphenylether made by J. T. Baker), ORTHO X-77 (a mixtureof fatty acids, fatty alcohols and isopropanol made by Chevron ChemicalCompany), Sweep 4F (chlorothalonil from Diamond Shamrock Company),Kinetic® (a proprietary blend of polyalkyleneoxide modifiedpolydimethylsiloxane and (nonionic surfactant) polyoxypropylene blockcopolymers made by Helena Chemical Company). These or similar agents maybe added to the aqueous solution to aid in seed treatment. Appropriatepenetrating agents can also be added to the solution to increasepenetration of the bioregulator compound. These penetrating agents couldinclude, but are not limited to: B-cyclodextrin(B-(heptamer)-cyclodextrin made by Takeda Chemical Industries, Ltd.) orTween (polyoxyethlene (20) sorbitan monooleate, available from E. Merck,Darmstadt, Germany).

C. Methods for Seed Application

1. Seed Soaking

In a preferred embodiment of the present invention, the bio-regulatorcompositions according to this invention are applied to seeds in theamount of about 0.001 mg to 0.5 mg of active bio-regulator ingredientper seed via bio-regulator solution. Bio-regulator solution is made bydissolving the composition to be used into a solution(solvent—preferably water, surfactants, and other appropriate wettingagents and penetrating adjuvants) at a concentration of preferably 0.05to 200 ppm. When using soaking as the method of bio-regulator solutionapplication, seeds should be preferably soaked for one quarter hour to24 hours. Other methods of application to seeds such as encapsulation,gel-coating, spraying, coating, dusting, seed priming and the like withthe bio-regulator compositions can be accomplished according toconventional methods, and are encompassed and contemplated by thisinvention

In other embodiments, the bio-regulatory effective amount of thecomposition to be applied may vary depending upon the seed species andcultivar and the desired overall effect. Generally, to achieve growthenhancement, treatment to seeds prior to the stages of plant developmentis preferred. The degree of penetration of the bio-regulator compositioninto the seed will be a factor in the overall growth enhancementachieved, and is dependent in part upon whether or not one or morepenetrating agents are used.

2. Seed Coating

In another embodiment of the invention, the process of seed coating isemployed. Seed coating involves application of certain activeingredients, including but not limited to fungicides, insecticides,plant growth promoting compounds, beneficial microbes, to seeds with abinder and other inert materials to facilitate mechanical planting ofseeds and protect the seeds once they are planted.

The process of seed coating generally is well-known in the art. It isoften achieved as pharmaceutical coating is achieved (i.e. throughapplying a mist, then an inert powder, and finally a binder). When thebio-regulator solutions or bio-regulator compositions are incorporatedinto a seed coating process, the desired plant growth enhancement can beachieved.

Application of bio-regulator solution or bio-regulator compositions canbe achieved when the seeds are misted with solution (preferably water)by adding the bio-regulator solution to the applied mist in similarconcentrations as compared to soaking. Application can also be achievedby applying a mixture of powderized bio-regulator compositions and inertpowder after the misting stage (crystalline, or inert particulatematerials impregnated with bio-regulator). Again, the concentrationsused when incorporating a powderized form of bio-regulator compositionwill be similar to the concentrations used when soaking (i.e. 0.05 to200 ppm or 0.00002 mg to 0.5 mg per seed).

In another embodiment, bio-regulator solutions can also be used withfungicides and insecticides. The bio-regulator solution can betank-mixed with most seed protection products provided such products aremiscible in water and labeled for slurry application directly on seed.Alternatively, the bio-regulator may not need to be mixed and misciblein water if applied by appropriate spray systems. Compatibility can bechecked by mixing a small amount of each product together to confirmsuitability of slurry application prior to application.

In another embodiment, the bio-regulator solutions can also be used withbiological products. They can be sequentially or simultaneously appliedwith most biological products when mixed in separate mix tanks orapplied by direct tank mixing the bio-regulator solution in the sametank with biological products such as legume inoculants, beneficialfungi, and other live microorganisms.

In this embodiment the method is preferably used to enhance or inhibitseed metabolic activity that is made amenable to bio-regulator compoundactivity as a function of the metabolic status achieved and maintainedduring or following the seed priming process to influence seedgermination and derived plant growth and development under a range ofenvironments that include the presence and absence of abiotic and/orbiotic stress.

D. Method for Application During Stages of Plant Development

Although seed treatment prior to the stage of plant development ispreferred, in other embodiments of the present invention, treatment mayoccur at any stage of plant development. When application is made to theseedling, or at the cotyledon, true leaf, two-leaf or four-leaf stages,etc., the preferred treatment includes about 0.0001 mg to 0.3 mgbio-regulator ingredient per plant. This can be accomplished by using atreatment rate of about 0.1 to about 200 ppm and preferably from about 1to about 120 ppm. Application according to this method preferablyfollows the emergence of the first true leaf and thinning, if thinningis necessary. Then, when the vegetative plant begins to develop, thebio-regulator solution may be sprayed, dusted, or otherwise applied tothe plant at a particular stage of development or at multiple stages ofthe plant's development. Application during stages of plant developmentis in embodiments directed toward influencing efficacious production ofparticular metabolic end products, plant yield, abiotic and/or bioticstress tolerance or pest resistance, or accelerating and enhancingherbicide activity in a plant burndown application.

Generally, to achieve increased herbicide phytotoxicity and plant killrate (i.e. burndown), spray treatment with the bio-regulator included ata desired concentration into the herbicide spray composition is thepreferred method. In at least one exemplary embodiment, plants treatedwith a combination of a herbicide and a bio-regulator according to oneembodiment of the present invention were killed 3 to 5 days faster ascompared to herbicide alone. A person skilled in the art may appreciatethat use bio-regulation to increase herbicide phytotoxicity may reducethe overall need for herbicide, and thereby reduce the environmental andeconomic impact of such herbicides.

E. Method for Application to Plants That Asexually Propagate

In another embodiment of the present invention, methods for applicationto asexually propagating plants are considered. There are a variety ofplants capable of asexual propagation. Cuttings are the parts of plantssevered from the parent to initiate the propagation. Once the cuttingsare formed, they may be rooted or grafted using a rooting medium.Rooting is the preferred time for treatment with the bio-regulatorysolution. However, the bio-regulatory solution can also be applied afterrooting and during transplantation, or during subsequent irrigation.Transplanting is done by means generally known to those skilled in theart. The cuttings and graftings are then irrigated and given therequisite amount of sunlight for maximum growth.

The bio-regulator solutions used to treat the cuttings are formed fromthe groups of compositions from the groups mentioned above (i.e. BMTA,BMVE, and BMIA), water, and appropriate wetting agents and penetratingadjuvants.

1. Hypothesis and Theory

Without any intention of limiting the scope of the invention, it istheorized that the bio-regulatory and stimulatory compositions used inthe method of this invention play a role in the physiologicaldevelopment of the plant by conferring beneficial enhancements from theseed stage and throughout a plant's full life cycle. This theory isformulated after observing that test plants are found to achieve variousbeneficial characteristics, which increase the plant biomass, whileuntreated, control plants, which were grown under similar conditions asthe test plants, did not exhibit these same growth enhancements. Thistheory is also furthered by the chemical compositions and formulationsused in this method, and the known and unknown effects thesecompositions and formulations have on plants at the physiological andmolecular level. It should also be noted that all compositions are notactive for all species but each of the plants tested did result in someform of enhancement via a combination of the bio-regulatorycompositions.

Furthermore, similar biological mechanisms may be involved in activatinggene expression in a manner that renders particular plants moresusceptible to a burndown herbicide application when the bio-regulatorcompounds are included in the herbicide mix and applied to the plants inappropriate concentrations. For example, a bi-regulatory activeconcentration of a composition according to the present invention maymodify the expression of a gene or genes that may be detrimental to thegrowth of an undesirable plant in a given environment.

F. Genetic Expression Studies

Modifications to gene expression in seeds and plants can occur whenseeds and plants are exposed to particular ecological and chemicalfactors, including abiotic and/or biotic stresses, plant growthpromoting compounds, systemic acquired resistance (SAR) promotingcompounds, and various pesticide compounds, including fungicides,insecticides and herbicides. Modifications to gene expression in seedsand plant can vary according to the ecological and/or chemical factor towhich the seed or plant is exposed. For example, when seeds and plantexperience abiotic or biotic stresses, mRNAs transcribed from acomplement of genes activated as part of a stress response produceproteins that aid in protecting the against the particular abiotic orbiotic stress experienced by the seed or plant; if the stress is hightemperature, the proteins expressed serve in protecting against injuryfrom high temperatures. Similarly, seeds and plant express certain geneprotein products in response to SAR promoting compounds that play rolesin protecting against pathogens. Further, seeds and plant express geneprotein products in response to exposure to plant growth promotingcompounds that play roles in acceleration of growth and developmentand/or increased growth in the presence or absence of abiotic and/orbiotic stresses. Proteins expressed in seeds and plant in response toexposure to certain pesticide compounds may or may not augment thefunction of the particular pesticide compounds. Plant phenotypesresulting from the interaction of seeds and plants with such ecologicaland chemical factors is a function of how gene expression is influencedby such factors.

Given the enhanced phenotypes produced in the absence and presence ofabiotic and biotic stresses as a function of treatment of seeds andplants with the bio-regulators, one object of the studies of embodimentsof the present invention set forth herein was to characterize geneexpression in seeds and plant treated with different bio-regulatorcompound compositions according to the present invention. Accordingly, aseries of studies were conducted of embodiments of the present inventionin order to illustrate the effect compositions of a model bio-regulator,BMVE, have on gene expression in monocotyledonous and dicotyledonousplant species. The studies were conducted on the dicotyledonous plantspecies, Arabidopsis and soybean and the monocotyledonous plant species,maize.

Gene expression analyses involved extracting mRNA from leaves of plantsgrown in a greenhouse under non-stress conditions approximately 15 hoursfollowing a foliar spray application of 3 ppm BMVE solution, andextracting mRNA from leaves of plants grown in a greenhouse undernon-stress conditions and not treated with the BMVE solution (controlplants). The extracted mRNA products were then analyzed by microarrayanalysis in the case of Arabidopsis and maize, and microarray andIllumina analyses in the case of soybean. The analyses identified whichgenes were either “up-regulated” or “down-regulated” by the BMVEtreatment based on comparing quantities of particular gene productsextracted from the plants treated with BMVE to the corresponding geneproducts extracted from the baseline control plants. Up-regulated geneswere increased due to the BMVE treatment, and down-regulated genes weredecreased due to the BMVE treatment. Once up-regulated anddown-regulated genes were identified in each of the plant species bymicroarray analysis, the genes were compared to genes in known genedatabases for each species to determine the function for each gene.

In one study of an embodiment of the present invention, the geneexpression profile of Arabidopsis plants treated with BMVE was comparedto the Arabidopsis eFP Browser database gene expression profiles forArabidopsis plants exposed to certain chemical applications, hormoneapplications, biotic stresses, and abiotic stresses. The microarray andcomparison results indicated that genes involved in protecting plantsfrom abiotic and biotic stresses and facilitating general growth anddevelopment were influenced by the BMVE treatment. There were 127 genesup-regulated by the BMVE treatment, including pathogen defense responsegenes, JA-response genes, Lipoxygenases, chitin-responsive proteins,dehydrin proteins, WRKY transcription factors that are particularlyinvolved in plant persistence in diverse abiotic and biotic stressenvironments, and root development genes. The plants treated with BMVEgenerally expressed 54 down-regulated genes including auxin-responsiveproteins and transcription factors.

In another study of an embodiment of the present invention, soybeanplant microarray and Illumina gene expression analyses were also studiedin a similar fashion as compared to the Arabidopsis study and the studyindicated that genes involved in protecting plants from abiotic andbiotic stresses and facilitating general growth and development wereinfluenced by treatment with BMVE. The result of the soybean plantmicroarray showed that there were 645 genes affected by the BMVEtreatment. Up-regulated genes included pathogenesis-related and pathogendefense genes. Down-regulated genes included proteinase inhibitors(PR-6) and WKRY transcription factors.

In another study of an embodiment of the present invention, maize plantswere treated with BMVE. The results generally indicated that there werea smaller number of genes affected in maize than in the soybeanmicroarray analysis. The particular genes influenced by treatment withBMVE were not identified due to limitations of the maize genomedatabase.

In these studies, plants were used, instead of the embryonic axis ofseeds, for example, because of the practicality of extracting mRNA fromlarge quantities of leaf tissue as contrasted to smaller quantities ofseed embryo tissue. Outside of genes associated with particular plantdevelopmental stages, it is expected that, within species and cultivar,influences of the bio-regulator compound, BMVE, on genes involved inprotecting plants from abiotic and biotic stresses and facilitatinggeneral growth would be similar at the seed embryo through mature plantstages of development. Further, in these studies, compound BMVE wasinvestigated as a model bio-regulator compound in order to help definemechanisms associated with the improved phenotypes resulting frombio-regulator treatment of seeds and plants prior to and during growthin the presence and absence of abiotic and biotic stresses. Givenphenotypes within a plant species and cultivar vary in either anon-stress or abiotic and biotic stress environment according to thebio-regulator compound used in treatment, it is expected that geneexpression profiles within a plant species and cultivar will vary inaccordance with the bio-regulator compound used in treatment.

A person skilled in the art may appreciate that the bio-regulatoryeffects produced by compositions according to embodiments of the presentinvention may not be limited to plant organisms. Gene expression infungi and algae may also be subject to bio-regulation. In at least oneembodiment of the present invention, microorganisms exposed to treatmentwith a bio-regulator according to embodiments of the present inventiondemonstrated modified gene expression. Furthermore, such modified geneexpression persisted generationally even when no bio-regulator waspresent. In one example, such modified gene expression persisted for twogenerations.

Bio-regulation of microorganisms may include modifying gene expressionof beneficial microorganisms to enhance their growth and vitality.Conversely, bio-regulation of microorganisms may include modifying geneexpression of harmful microorganisms to suppress them or make them moresusceptible to other biological or chemical agents.

G. EXAMPLES

Studies of embodiments of the present invention encompassing varioustypes of plants and seeds with various concentrations of appliedbio-regulatory compositions have further illustrated the effectivenessof the bio-regulator compositions, solutions, and the applicationmethods and procedures contained herein. In these embodiments, testplants that have been treated with the bio-regulatory compositions werecompared to control plants. Each of the groups of plants is grown undersimilar environmental conditions. After different lengths of time, testplants are compared to control plants, considering both internal andexternal physical characteristics. These studies took place in variousgeographic locations including Malaysia, California, Nebraska, Kansas,and Oklahoma.

1. General Procedure for Seed Soaking Treatment for Growth Enhancementof Agricultural Crops and General Flora:

In this preferred embodiment of the present invention, seeds are soakedin prepared bio-regulator solutions for a period of one half to fourhours depending on the absorption property of the seeds. Thebio-regulator solutions are prepared in concentrations ranging from 0.1ppm to 100 ppm using compositions selected from the BMIA Compounds orEster Compounds. Because size and absorption characteristics of seedsvary, the soak time for each seed will also vary. For example, aparticular cotton seed may take at least four hours for good uptake ofbio-regulator into the seed. At the end of the soaking period thesolution is drained and while still wet, the seeds are planted intostarter potting soil or directly into soil. Rather than directlyplanting the seeds, they may be dried for planting at a later point.After germination seedlings are transplanted to larger pots or to opensoil.

a. Specific Examples Example I General Procedure for Seed SoakingTreatment of Agricultural Crops using Rice Seeds

In another preferred embodiment of the present invention, seeds aresoaked in prepared bio-regulator solutions for a period of three hours.Rice seeds were selected from ‘M-202’ medium-grain rice (Oryza sativaL.), (Reg. no 72) PI494105 type grains; this selection was meant torepresent a typical California rice seed, and was not meant to belimiting. The bio-regulator solutions are prepared using 5 ppm BMTAsolution with 0.05% v/v Kinetic® made by Helena Chemical Company. Thesolution was drained and the seeds planted in starter plastic containerswith potting soil. After germination and having grown to 4 inches inheight, the plants were transplanted into 6 inch diameter plasticcontainers. Two sets of three pots each were set up for this study; oneset for the test group and one set for the control group.

Both the control and test studies were irrigated with 1500 ppm NaClsolution from time of planting until the study was completed. Thepurpose of the NaCl irrigation was to determine the effect of thebio-regulator treatment in regards to salt tolerances.

It was observed that the BMTA treated rice plant showed enhanced growthfrom the time of planting, through completion of the study. The planthad observable increased biomass, tillering, and foliage.

In this embodiment, the effect of the bio-regulator solution on riceseed (Tables 1-A and 1-B) was studied. Those skilled in the art willrecognize that the effects, results and outcomes may be achievablefollowing a similar procedure for other types of cereal grains andmonocot plants.

This example is summarized in Table 1-A below, where Set 1 is thecontrol, and Set 2 is the test group. The results are summarized inTable 1-B:

TABLE 1-A Set # Treatment Ppm Soak Time Irrigation Surfactant Set 1 Tapwater 0 3 hours NaCl soln. Kinetic ® Set 2 BMTA soln. 5 3 hours NaClsoln. Kinetic ®

TABLE 1-B Results Biomass Height Tillering Set 1 Average Average AverageSet 2 ~3 x Set 1 Same ~3 x Set 1

Example II

In this preferred embodiment of the present invention, seeds are soakedin prepared bio-regulator solutions for a period of 24 hours. Rice seedswere selected from the same ‘M-202’ type grains as in the previousstudy. The bio-regulator solutions were prepared using 5 ppm BMPAsolution. The solution was drained and seeds planted in starter plasticcontainers filled with potting soil. After germination and plant growthreaching height of four inches, rice seedlings were transplanted fromtheir greenhouse containers to six-inch diameter plastic containers andplaced outdoor. Again, two sets of three pots for the control and testgroup were used.

It was observed that the BMPA treated rice plant showed enhanced growthfrom the time of planting, through completion of the study. The planthad observable increased biomass, tillering, and foliage. This methodand bio-regulator solution produced greater growth enhancement than theprevious example's method and procedure.

These studies were also carried out using an increased amount of BMPA.The amount was increased from 1 ppm to 5 ppm, and the results of thestudy were similar to the results outlined in the paragraph above.

The study of this embodiment is summarized in Tables 2-A and 2-B below,where Set 1 is the control, and Set 2 is the test group. The results aresummarized in Tables 2-C and 2-D:

TABLE 2-A Set # Treatment Ppm Soak Time Irrigation Surfactant Set 1Water 0 24 hrs Tap water Kinetic ® Set 2 BMPA 5 24 hrs Tap waterKinetic ®

TABLE 2-B Set # Treatment Ppm Soak Time Irrigation Surfactant Set 1Water 0 24 hrs Tap water Kinetic ® Set 2 BMPA 1 24 hrs Tap waterKinetic ®

TABLE 2-C Results Biomass Height Tillering Set 1 Average Average AverageSet 2 ~4 x Set 1 ~1.2 x Set 1 ~4 x Set 1

TABLE 2-D Results Biomass Height Tillering Set 1 Average Average AverageSet 2 ~4 x Set 1 ~1.2 x Set 1 ~4 x Set 1

Example III General Procedure for Treatment of Floral Plant Cuttings inGeneral and Geranium Slips in Particular

In this preferred embodiment of the present invention, according tocommon geranium cutting means, geraniums were slipped into 18 slips ofsimilar length. In this example the slips had no flower buds. The slipswere separated into four different sets: three slips each in three ofthe sets, and then nine slips in the final set. The slips are thentreated with bio-regulator solutions formed from compositions from thedifferent groups and water. In the first set, the slips were treatedwith BMVE solution; in the second set, the slips were treated with BMTAsolution; in the third set, the slips were treated with BMIA in bromidesolution; and, in the fourth set, the nine slips were treated withwater.

The treatment with bio-regulator solution preferably occurs in theinitial planting of the geranium slips, but can also occur at any timefrom the forming of the geranium slips up to and including thetransplanting of the slips. The treatment can occur after transplanting,but this is not the preferred method.

Transplanting can be done by means generally known to those skilled inthe art. In this example the slips were transplanted into 6-inchdiameter pots and filled with potting soil. Each of the pots wereidentified as belonging to the first, second, third, or fourth set. Eachpot, after transplantation, was thoroughly irrigated with water. Eachset of pots were placed outside for maximum sunlight, for eight hoursper day.

It was observed that the treated geranium slips generally showedenhanced growth from the time of transplanting, through completion ofthe study. The slips had observable increased vigor even when comparedwith slips that had a greater number of leaves.

In this embodiment, the effect of bio-regulator solutions on geraniumslips was studied; however, it is anticipated that similar effects,results, and outcomes will be achievable following a similar procedureas outlined above for other types of plants that reproduce throughvegetative propagation.

The study associated with this embodiment is summarized in Table 3-Abelow, where Set 1 is the control group and Set 2 is the test group. Theresults are summarized in Table 3-B. Included for convenience is Table3-C, which summarizes the total test heights to control heights ratios.The ratios for Table 3-C are calculated by totaling the heights grown byeach slip in each set and comparing it to the control counterpart thatwas grown in similar environmental conditions, without the treatment ofthe bio-regulator solutions. The total heights grown by the test slipsare also compared to the control slips by a percentage of the overallheight of the control slips.

TABLE 3-A Depth Planted Set # Number of Slips Treatment (in.) Set 1 3BMVE 2½ Set 2 3 BMTA 2½ Set 3 3 BMIA 2½ Set 4 9 Water 2½

TABLE 3-B Set PGS Dosage Slip 1 Slip 2 Slip 3 Day # Treatment (ppm) Ht.(in.) Ht. (in.) Ht. (in.) 11 1 BMVE 5 2¼ 2⅞ 3⅜ 4 Water 2⅛ 2⅜ 2¼ 2 BMTA 52¾ 2¾ 3⅛ 4 Water 2⅛ 1¼ 1¼ 3 BMIA(Br) 5 3 1⅞ 3½ 4 Water 1⅜ 2⅝ 2¾ 27 1BMVE 5 2⅜ 4¼ 3⅞ 4 Water 3¼ 2⅜ 2⅜ 2 BMTA 5 3⅛ 3¾ 3⅞ 4 Water 2½ 1¾ 2⅜ 3BMIA(Br) 5 3⅛ 3¼ 4⅛ 4 Water 3½ 3⅛ 3⅛

TABLE 3-C Sum (Slip Ratio: Day Set # Treatment 1 + 2 + 3) Test/Control %of Control 11 1 BMVE 8½  8.5/6.75 125.93% 4 Water 6¾ 2 BMTA 8⅝8.625/4.625 186.49% 4 Water 4⅝ 3 BMIA(Br) 8⅜ 8.375/7.75  108.06% 4 Water7¾ 27 1 BMVE 10½ 10.5/8   131.25% 4 Water 8 2 BMTA 10¾ 10.75/6.625162.26% 4 Water 6⅝ 3 BMIA(Br) 10½ 10.5/9.75 107.69% 4 Water 9¾

Example IV General Procedure for Bulb Soaking Treatment using HollandNarcissus Bulbs

In this preferred embodiment of the present invention, bulbs are soakedin prepared bio-regulator solutions for two different periods. The firstperiod was two hours. The second period was three and three quarterhours.

The bulbs were selected on November 25, from packets of Dutch Mastertype bulbs procured from Van Bioem Gardens, located in Meridan,Mississippi, which bulbs are a product of Holland. This selection wasmeant to be representative of general bulb-type plants and was not meantto be limiting. The selected packet originally contained 26 bulbs,however, the three largest and five smallest were removed and discardedso that the study would only encompass approximately average-sizedbulbs. The remaining 18 bulbs were separated into three sets with sixapproximately equal-sized bulbs per set.

Two of the sets of six bulbs (12 in total) were placed into one sealablecontainer with 20 ounces of bio-regulator solution, which was composedof 5 ppm BMVE and 0.05 ppm Kinetic®. The remaining six bulbs were placedinto a second sealable container with 10 ounces of water. Both of thecontainers were rotated every five to 10 minutes in order to allow foreven distribution and absorption of the bio-regulator solution and waterrespectively.

As mentioned above, after two hours the first set of six bulbs wereremoved from the container. These bulbs were drained and air-dried. Oneand three quarter hours later the remaining bulbs were removed from bothcontainers and air-dried. All sets were planted into marked clay potscontaining potting soil and subsequently irrigated. The first set ofbulbs that soaked for two hours were marked Set 1. The second set ofbulbs that soaked three and three-quarter hours were marked Set 2. Thebulbs that soaked in water for three and three-quarter hours were markedas the Control group.

The pots were six-inches in diameter and five-inches high. The pots werethen placed outdoors and irrigated with water as needed, or according toknown practice in the art.

On January 17, bulb shoots began emerging with green foliage. On March1, measurements were taken of the height of the tallest leaf stalk ofeach plant (the stalks without a flower). Also, on March 1, measurementswere taken of the stalks with flowers, which were found on sevendifferent plants (each of these seven different plants may or may nothave had multiple measured stalks). Each stalk that was measured wasnumbered.

In the study of this embodiment, it was observed that the BMVE treatedbulbs showed enhanced growth from the time of planting, throughcompletion of the study. The stalks had observable increased biomass.The enhanced growth observed possibly could have been even moresubstantial had it not been for “twinning” (where more than one plant isattached) that was observed among the treated bulbs.

This example is summarized in Table 4-A, Table 4-B, and Table 4-C below.

TABLE 4-A (Control treated with water, Set 1 treated with BMVE-twohours, and Set 2 treated with BMVE-three and three-quarter hours):Measurement of Stalks That did not Have Flowers Stalk No. Control Set 1Set 2 1 6 10.25 7.5 2 5.75 6.625 6.875 3 5.5 8.875 4.875 4 4.5 7.1256.375 5 3.125 7.5 6.375 6 2.1875 4.5 6.125 7 1.5 6.625 4.75 8 1.5 6.5 69 6.5 4.125 10 4.625 5.625 11 6 5.25 12 4.5 4.75 13 4.125 4.625 14 3.3754.625 15 7.5 Total 30.0625 94.625 77.875 Avg. Ht. 3.76 6.31 5.56

TABLE 4-B Measurement of Stalks without Flowers and Stalks with FlowersMeasurement of Stalk Ht. Measurement of Stalk Ht. with Flower Stalk No.Control Set 1 Set 2 Stalk No. Control N1 N2 1 9.5 1 8.25 12.65 2 6.62511.625 9 2 3 8.875 3 7.875 10.875 4 5.5 11.375 4 8.75 5 8.625 5 7.759.625 6 6 8.125 6 8.75 7 6.5 9.25 5.75 7 8 6.375 8 8.875 8.375 9 7.258.375 9 5 10 2.625 6.75 10 8.25 11 3.875 8.5 8 11 12 3.875 7.875 12 8.513 3.5 7.5 13 7.5 14 8.5 6.25 14 15 6.75 7.375 15 16 5.125 6.5 16 Total44.875 90.75 86.125 Total 28.88 50.5 41.625 Avg. Ht. 4.99 8.25 7.83 Avg.Ht. 7.22 10.1 8.325

TABLE 4-C Percent of Control (from March 6) Measured Stalks that didMeasured Stalks that had no Flower have Flower % of % of No. Avg. Con-No. Avg. Con- Stalks Ht. trol Stalks Ht. trol Con- 9 4.99 Con- 4 7.22trol trol Set 1 11 8.25 165% Set 1 5 10.1 140% Set 2 11 7.83 157% Set 25 8.325 115%

Example V General Procedure for Seed Soaking Treatment of AgriculturalCrops Using Radish Seeds

In this preferred embodiment of the present invention, seeds were soakedin prepared bio-regulator solutions for a period of three hours. Radishseeds were selected from a local nursery (September 28). This selectionwas not meant to be limiting but was meant to be a selection of atypical California radish seed. Two bio-regulator solutions wereprepared. The first bio-regulator solution was prepared using 5 ppm BMVEsolution combined with 0.05% v/v Kinetic®. The second bio-regulatorsolution was prepared using 3 ppm BMIA in water and Kinetic® solution.The solution was drained and the seeds planted in marked clay pots withpotting soil. The clay pots were four inches high and six inches indiameter. Three sets of two pots were set up for this study; one set forthe test group with BMVE treatment, one set for the test group with BMIAtreatment and one set for the control group.

On January 1, a visual review of all of the radish tops was conducted.It was observed that all three sets of plants were very close in vigorand chlorophyll green. Then the radish plants were removed from claypots for root evaluation. The treated plants showed an increased numberof fine roots as compared to the control plants. The radish plants wereclipped, cleaned and weighed for a more accurate determination oftreatment effects.

The results showed that the treated radish plants had increased biomass.The study of this embodiment is summarized in Table 5-A below, and theresults are summarized in Table 5-B.

TABLE 5-A Soak Time Treatment PPM (hrs) Surfactant Location Water 0 3Kinetic ® Outdoor BMVE 5 3 Kinetic ® Outdoor BMIA 3 3 Kinetic ® Outdoor

TABLE 5-B Treatment Weight (gm) Water 11 BMVE 17 BMIA 12

Example VI General Procedure for Seed Soaking Treatment of AgriculturalCrops Using Lemon Seeds

In this preferred embodiment of the present invention, seeds were soakedin prepared bio-regulator solutions for a period of three hours. Lemonseeds were selected from Eureka Lemon Tree (January 5). This selectionwas not meant to be limiting but was meant to be a selection of atypical lemon seed. The lemon seeds were separated into three sets withseven seeds in each set The first set, Set 1, was the control set, andwas soaked in water with Kinetic® surfactant. The second set, Set 2,soaked in BMVE solution with Kinetic® surfactant. The third set, Set 3,soaked in BMIA solution with Kinetic® surfactant. The seeds were driedand planted.

The seeds were observed until sprouts appeared. When sprouts began togrow, sprouts were selected from each pot with lengths approximatelythree inches long. These sprouts were transplanted to pots that weresix-inches in diameter, and seven inches tall. These pots were placedoutdoors. A month after the initial planting, the test study wasduplicated.

The results showed that the treated plants had increased roots, sprouts,and overall biomass as compared to the control. The study of thisembodiment is summarized in Table 6-A, and in Table 6-B. The overallresults are summarized in Table 6-C.

TABLE 6-A Treatment PPM Soak Time Set 1 Water 0 3 hrs Set 2 BMVE 5 3 hrsSet 3 BMIA 3 3 hrs

TABLE 6-B Set # Date Treatment Sprouts Description Set 1 24 Feb Water 0Set 2 24-Feb BMVE 0 Set 3 24-Feb BMIA 2 5 and 12 mm long Set 1 1-MarWater 2 5 and 5 mm long Set 2 1-Mar BMVE 0 Set 3 1-Mar BMIA 3 sproutswith roots Set 1 4-Mar Water 3 sprouts with roots Set 2 4-Mar BMVE 0 Set3 4-Mar BMIA 7 sprouts with roots

TABLE 6-C % of % of Control Control Date Planting Control BMVE BMIA(BMVE) (BMIA) 22-Aug 1st 5.125 8.625 7.5 168% 146% 22-Aug 2nd 4.8755.875 6.375 121% 131% 20-Sep 1st 7.75 12.125 9.875 156% 127% 20-Sep 2nd7.5 8.125 9 108% 120% 21-Nov 1st 8.5 16 13.75 188% 162% 21-Nov 2nd 9.7511.5 12 118% 123%

Example VII General Procedure for Seed Soaking Treatment of AgriculturalCrops Using Cotton Seeds

In this preferred embodiment of the present invention, seeds were soakedfor a period of 3 hours in the bio-regulator solution. Cotton Bt wereselected and studied to determine if bio-regulator chemistry willenhance or influence the inserted Bt genes of the transgenic plant. Theseeds were separated into two different sets on July 2. The first set,Set 1, was soaked in 5 ppm BMPA solution with 0.05% Kinetic®. The secondset, Set 2, was soaked in water with 0.05% v/v Kinetic®.

The solution was drained and the treated seeds planted in starterplastic trays. After germination and after the seedlings reached theheight of four inches, the cotton Bt plants were transplanted intolarger six-inch plastic containers. The cotton plants were placedoutdoors for the duration of the study. The different sets of plantswere regularly observed throughout this study.

After 47 days from planting, it was noted that the underside of the leafof the untreated cotton Bt plant showed traces of aphid infestation. Notrace of aphid was observed on the treated cotton Bt plant, which wasplaced 20 inches from the untreated cotton Bt plant.

After 55 days from planting, it was noted that the aphid infestation ofthe untreated cotton Bt plant was heavy and the plant was beginning toshow stress. No aphid was found on the treated cotton Bt plant. After 57days from planting, it was noted that a few aphids were detected on thetreated plant. After 74 days from planting, it was noted that thetreated plant appeared to be infested with aphid and showing somestress.

For the study of this embodiment, the treatment using the bio-regulatorchemistry (BMPA) appears to delay the infestation of aphids on thecotton Bt plant by as much as 19 days. The study for this embodiment issummarized in Table 7-A below, and the results are summarized in Table7-B.

TABLE 7-A Treatment PPM Soak Time Surfactant Set 1 Water 0 3 hrsKinetic ® Set 2 BMPA 5 3 hrs Kinetic ®

TABLE 7-B Days After Planting Control Treated 47 L-Aphid No Aphid 55H-Aphid No Aphid 57 H-Aphid L-Aphid 74 Stressed M-Aphid Key: L—lightinfestation; M—medium infestation; H—heavy infestation;Stressed—overcome by infestation

In this example, the effect of the bio-regulator solution on cotton seedwas studied. However, it is anticipated that similar effects, results,and outcomes will be achievable following a similar procedure asoutlined above for plants in the Gossypium genus.

Example VIII General Procedure for Treatment of Agricultural Crops UsingSoybeans Spray Tests

In this preferred embodiment of the present invention, three seeds eachof soybean varieties Thorn (non-Roundup Ready variety) or S30-D4(Syngenta Roundup Ready variety) were sown in 6 inch pots (50 total potswith 3 seeds each per variety) containing soybean potting mix (40%Canadian Peat, 40% Coarse Vermiculite, 15% Masonry Sand, 5% ScreenedTopsoil, 3.63% Waukesha fine lime, 0.46% Micromax, 0.68% AquaGro, and0.23% Hi Yield Iron Plus). Pots were placed in the greenhouse growthregime initially set at 29° C., 14-hour days and 24° C., 10-hour nights(23 days after planting (DAP), the growth regime was set at 25° C.,14-hour days and 15° C., 10-hour nights). Plants were watered on Monday,Wednesday, and Friday. The plant irrigation schedule involvedalternating plain water, 500 ppm Ca, and 350 ppm N and 60 ppm Fe.Following emergence and cotyledon expansion, plants were thinned to oneplant per pot. A significant number of the Thorn soybean plants derivedfrom the seeds provided by UNL Beadle Center were abnormal and, overall,the plants showed a significant lack of uniformity with regards todevelopment. Therefore, work on the Thorn soybean plants wasdiscontinued. S30-D4 soybean plants were sprayed with 1 or 3 ppm BMVE in0.05% Tween 20 or 0.05% Tween 20 (control) at the first trifoliate leafstage, the fifth trifoliate leaf stage and the R1 (flowering) stage ofdevelopment (3, 7 and 10 sprays per plant to ensure complete plantcoverage at the first trifoliate, fifth trifoliate and flowering stages,respectively). At 34 DAP, five of the ten replications of soybean plantspreviously treated with foliar applications of the control, BMVE 1 ppm,and BMVE 3 ppm treatments at the first and fifth trifoliate leaf andflowering stages were irrigated at a rate of 1 L per pot with 100 mM ofNaCl in either 350 ppm N and 60 ppm Fe (Monday), water (Wednesday), or500 ppm Ca (Friday), while the remaining 5 replicas were irrigatedsimilarly except that NaCl was excluded. At flowering plant leaf tissuefrom saline stressed and non-stressed plants were sampled for extractionof RNA to be used in Illumina and/or microarray analyses.

Over the course of soybean plant development, measurements were made toassess phenotypes as related to the foliar treatments. Plant height,leaf expansion, internode length, rate of photosynthesis, leafchlorophyll content, and flowering and pod production time data areprovided below in the Results section.

Seed Soak Tests

In another preferred embodiment of the present invention, SyngentaS30-D4 soybean seeds were soaked in water or 0.05% Tween 20 (controls)or BMVE at 1, 3, and 6 ppm in 0.05% Tween 20 and then planted in 6 inchpots containing soybean potting mix. Following emergence and expansionof the first trifoliate leaves, plants were thinned to one plant perpot. These experiments are currently ongoing.

Observations Spray Tests

Table 8-A shows mean height, leaf expansion, and internode length valuesof S30-D4 soybean plants following foliar applications with 0.05% Tween20 (control), or 1 or 3 ppm BMVE in 0.05% Tween 20 (BMVE 1 ppm and BMVE3 ppm, respectively). Ten replications of 4 S30-D4 soybean seeds wereplanted in 6 inch pots in the greenhouse at a growth regime initiallyset at 29° C., 14-hour days and 24° C., 10-hour nights (twenty-threedays after planting (DAP), the growth regime was set at 25° C., 14-hourdays and 15° C., 10-hour nights). Following full expansion of the firsttrifoliate leaf, seedlings were thinned to one plant per pot and theremaining 50 seedlings appeared uniform in size and development. At thefirst and fifth trifoliate leaf and at flowering, foliar control, BMVE 1ppm, and BMVE 3 ppm treatments were applied with a spray bottle (3, 7and 10 sprays per plant to ensure complete plant coverage at the firsttrifoliate, fifth trifoliate and flowering stages, respectively). Plantheights and leaf expansion measurements were taken at the fifthtrifoliate leaf stage and internode lengths were recorded at theinitiation of flowering.

In at least one embodiment where S30-D4 soybean plants received foliarapplications at the first and fifth trifoliate leaf stage of BMVE at aconcentration of 3 ppm in 0.05% Tween 20. The plants showed increasedplant height as compared to a control treated with 0.05% Tween 20.

TABLE 8-A Leaf Expansion in cm (middle lobe of the 5^(th) InternodeLength Plant Height trifoliate leaf) 30 DAP in cm (4^(th) to 5^(th) (cm)29 DAP L L W W node) 41 DAP Mean StDev Mean StDev Mean StDev Mean StDevcontrol 21.08 2.95 10.70 0.46 6.67 0.53 2.51 0.35 B 1 22.35 1.61 10.830.63 7.25 0.38 2.71 0.31 ppm B 3 24.77 2.01 10.86 0.53 7.11 0.37 3.410.45 ppm

Photosynthesis Rate

Table 8-B shows mean and standard deviation from the mean of rates ofphotosynthesis obtained by quantifying decrease of CO₂ over time insidea leaf chamber enclosing 6 cm² of leaf tissue from the middle lobe ofthe fifth trifoliate leaf of S30-D4 soybean plants with a LI-6400XTinfrared gas analyzer following two foliar applications with 0.05% Tween20 (control), or BMVE at a concentration of 1 or 3 ppm in 0.05% Tween 20(BMVE 1 ppm and BMVE 3 ppm, respectively). Ten replications of 4 S30-D4soybean seeds were planted in 6 inch pots in the greenhouse at a growthregime initially set at 29° C., 14-hour days and 24° C., 10-hour nights(twenty-three days after planting (DAP), the growth regime was set at25° C., 14-hour days and 15° C., 10-hour nights).

Following emergence and development of the first trifoliate leaf,seedlings were thinned to one plant per pot and the remaining 50seedlings appeared uniform in size and development. Upon full expansionof the first trifoliate and fifth trifoliate leaf, foliar control, BMVE1 ppm, and BMVE 3 ppm treatments were applied with a spray bottle (3 and7 sprays per plant to ensure complete plant coverage at the first andfifth trifoliate stages, respectively). Photosynthetic rates weredetermined with the LI-6400XT at the fifth trifoliate leaf stage at 29DAP.

TABLE 8-B Photosynthetic rate (decrease in CO₂ over time in chamberenclosing fixed leaf area - μmol CO₂/m²/sec) Treatment Mean StDevControl 16.7 1.4 B 1 ppm 17.0 0.9 B 3 ppm 15.9 2.2

Chlorophyll Content

Table 8-C shows mean and standard deviation from the mean of relativechlorophyll content measured with a Minolta SPAD 502 chlorophyll meteron the uppermost, fully expanded trifoliate leaf of S30-D4 soybeanplants following three foliar applications with 0.05% Tween 20(control), or 1 or 3 ppm BMVE in 0.05% Tween 20 (BMVE 1 ppm and BMVE 3ppm, respectively). Ten replications of 4 S30-D4 soybean seeds wereplanted in 6 inch pots in the greenhouse at a growth regime initiallyset at 29° C., 14-hour days and 24° C., 10-hour nights (23 days afterplanting (DAP), the growth regime was set at 25° C., 14-hour days and15° C., 10-hour nights). Following full expansion of the firsttrifoliate leaf, seedlings were thinned to one plant per pot and theremaining 50 seedlings appeared uniform in size and development. At thefirst and fifth trifoliate leaf stage and at flowering, foliar control,BMVE 1 ppm, and BMVE 3 ppm treatments were applied with a spray bottle(3, 7 and 10 sprays per plant for complete plant coverage at the firsttrifoliate, fifth trifoliate and flowering stages, respectively). At 34DAP, five of the ten replications previously treated with foliarapplications of control, BMVE 1 ppm, and BMVE 3 ppm treatments at thefirst and fifth trifoliate leaf and flowering stages were irrigated at arate of 1 L per pot with 100 mM of NaCl in either 350 ppm N and 60 ppmFe (Monday), water (Wednesday), or 500 ppm Ca (Friday), while theremaining 5 replicas were irrigated similarly except that NaCl wasexcluded. Chlorophyll contents were measured at 49 DAP.

TABLE 8-C Chlorophyll Content - Chlorophyll Content - No Saline StressStress Treatment Mean St Dev Mean St Dev Control 37.5 2.0 37.8 1.6 B 1ppm 37.3 1.9 38.3 1.3 B 3 ppm 37.7 1.5 39.5 1.6

Flowering Nodes

FIGs. 6 a and 6 b illustrates nodes at which flowering occurred inS30-D4 soybean plants following foliar applications with 0.05% Tween 20(control), or 1 or 3 ppm BMVE in 0.05% Tween 20 (BMVE 1 ppm (B1 in FIGs.6 a and 6 b ))and BMVE 3 ppm (B3 in FIGs. 6 a and 6 b ), respectively).Ten replications of 4 S30-D4 soybean seeds were planted in 6 inch potsin the greenhouse at a growth regime initially set at 29° C., 14-hourdays and 24° C., 10-hour nights (23 days after planting (DAP), thegrowth regime was set at 25° C., 14-hour days and 15° C., 10-hournights). Following full expansion of the first trifoliate leaf,seedlings were thinned to one plant per pot and the remaining 50seedlings appeared uniform in size and development. Upon full expansionof the first trifoliate, fifth trifoliate leaf and at flowering, foliarcontrol, BMVE 1 ppm, and BMVE 3 ppm treatments were applied with a spraybottle (3, 7 and 10 sprays per plant to ensure complete plant coverageat the first trifoliate, fifth trifoliate and flowering stages,respectively). Flowering data was recorded 35 DAP.

FIG. 7 shows nodes at which pod formation occurred in S30-D4 soybeanplants following foliar applications with 0.05% Tween 20 (control), or 1or 3 ppm BMVE in 0.05% Tween 20 (BMVE 1 ppm (B1 in FIG. 7) and BMVE 3ppm (B3 in FIG. 7), respectively). Ten replications of 4 S30-D4 soybeanseeds were planted in 6 inch pots in the greenhouse at a growth regimeinitially set at 29° C., 14-hour days and 24° C., 10-hour nights(twenty-three days after planting (DAP), the growth regime was set at25° C., 14-hour days and 15° C., 10-hour nights). Following fullexpansion of the first trifoliate leaf, seedlings were thinned to oneplant per pot and the remaining 50 seedlings appeared uniform in sizeand development. Upon full expansion of the first trifoliate, fifthtrifoliate leaf and at flowering (31 DAP), foliar control, BMVE 1 ppm,and BMVE 3 ppm treatments were applied with a spray bottle (3, 7 and 10sprays per plant to ensure complete plant coverage at the firsttrifoliate, fifth trifoliate and flowering stages, respectively). At 34DAP, five of the ten replications of soybean plants previously treatedwith foliar applications of the control, BMVE 1 ppm, and BMVE 3 ppmtreatments at the first and fifth trifoliate leaf and flowering stageswere irrigated at a rate of 1 L per pot with 100 mM of NaCl in either350 ppm N and 60 ppm Fe (Monday), water (Wednesday), or 500 ppm Ca(Friday) (pod fill data in panel B), while the remaining 5 replicas wereirrigated similarly except that NaCl was excluded (pod fill data inpanel A). Pod formation data was recorded 42 DAP.

Saline Stress

Table 8-G shows mean and standard deviation from the mean of plantheight measurements on saline stressed and non-stressed S30-D4 soybeanplants following three foliar applications with 0.05% Tween 20(control), or 1 or 3 ppm BMVE in 0.05% Tween 20 (BMVE 1 ppm and BMVE 3ppm, respectively). Ten replications of 4 S30-D4 soybean seeds wereplanted in 6 inch pots in the greenhouse at a growth regime initiallyset at 29° C., 14-hour days and 24° C., 10-hour nights (23 days afterplanting (DAP), the growth regime was set at 25° C., 14-hour days and15° C., 10-hour nights). Following full expansion of the firsttrifoliate leaf, seedlings were thinned to one plant per pot and theremaining 50 seedlings appeared uniform in size and development. At thefirst and fifth trifoliate leaf stage and at flowering, foliar control,BMVE 1 ppm, and BMVE 3 ppm treatments were applied with a spray bottle(3, 7 and 10 sprays per plant for complete plant coverage at the firsttrifoliate, fifth trifoliate and flowering stages, respectively). At 34DAP, five of the ten replications previously treated with foliarapplications of control, BMVE 1 ppm, and BMVE 3 ppm treatments at thefirst and fifth trifoliate leaf and flowering stages were irrigated at arate of 1 L per pot with 100 mM of NaCl in either 350 ppm N and 60 ppmFe (Monday), water (Wednesday), or 500 ppm Ca (Friday), while theremaining 5 replicas were irrigated similarly except that NaCl wasexcluded. Chlorophyll contents were measured at 49 DAP.

TABLE 8-G Plant Height (cm) - Saline Plant Height (cm) - No StressedStress Treatment Mean St Dev Mean St Dev Control 40.8 1.1 46.4 2.1 B 1ppm 44.6 3.6 54.0 2.5 B 3 ppm 44.2 3.4 58.0 8.6

Table 8-H shows mean and standard deviation from the mean of leaf areameasurements on saline stressed and non-stressed S30-D4 soybean plantsfollowing three foliar applications with 0.05% Tween 20 (control), or 1or 3 ppm BMVE in 0.05% Tween 20 (BMVE 1 ppm and BMVE 3 ppm,respectively). Ten replications of 4 S30-D4 soybean seeds were plantedin 6 inch pots in the greenhouse at a growth regime initially set at 29°C., 14-hour days and 24° C., 10-hour nights (23 days after planting(DAP), the growth regime was set at 25° C., 14-hour days and 15° C.,10-hour nights). Following full expansion of the first trifoliate leaf,seedlings were thinned to one plant per pot and the remaining 50seedlings appeared uniform in size and development. At the first andfifth trifoliate leaf stage and at flowering, foliar control, BMVE 1ppm, and BMVE 3 ppm treatments were applied with a spray bottle (3, 7and 10 sprays per plant for complete plant coverage at the firsttrifoliate, fifth trifoliate and flowering stages, respectively). At 34DAP, five of the ten replications previously treated with foliarapplications of control, BMVE 1 ppm, and BMVE 3 ppm treatments at thefirst and fifth trifoliate leaf and flowering stages were irrigated at arate of 1 L per pot with 100 mM of NaCl in either 350 ppm N and 60 ppmFe (Monday), water (Wednesday), or 500 ppm Ca (Friday), while theremaining 5 replicas were irrigated similarly except that NaCl wasexcluded. Leaf area measurements were estimated on the trifoliate leaffrom the seventh node with (SigmaScan Pro 5.0, Systat Software Inc.,Point Richmond, Calif.).

TABLE 8-H Leaf Area - Saline Stressed Leaf Area - No Stress TreatmentMean St Dev Mean St Dev Control 163.1 12.0 167.0 15.1 B 1 ppm 188.7 23.6186.7 14.6 B 3 ppm 191.0 6.9 172.1 17.1

3. Results and Conclusions:

Based on studies of these embodiments, foliar treatment of S30-D4soybean plants with BMVE at 1 and 3 ppm appeared to increase the rate ofplant growth and development in saline stress and non-stress regimes;increases were particularly notable at the 3 ppm rates. Based on themeasurements taken with the Minolta SPAD 502 meter and the Li-6400XTinfrared gas analyzer in this investigation, chlorophyll content likelywas not increased per unit area by the bio-regulator chemistries.However, an increase in total photosynthetic capacity per plant islikely given that overall vegetative matter/leaf surface area wasincreased.

Example IX General Procedure for Seed Soaking Treatment of AgriculturalCrops Using Corn Seeds

In a preferred embodiment of the present invention, corn seeds weretreated with 5 ppm BMVE. Corn seeds were treated and germinated in theMartinez, Calif., greenhouse. After seedlings reached heights of fourinches tall, seedlings were placed outdoors for the remaining period ofstudy. After seedlings reached 8 to 12 inches, they were exposed totemperatures exceeding 100 degrees Fahrenheit for several days (rangeswere from 100 to 115 degrees F.). The treated corn seedlings continuedhealthy growth under these conditions while the control seedlings didnot survive.

The results of the study of this embodiment showed that the treatedplants had increased roots, sprouts, and overall biomass as compared tothe control. The study that encompasses this example is summarized inTable 9.

TABLE 9 Dosage Temp Location Planted Photo Treatment (ppm) (degrees F.)Greenhouse Aug 26 BMVE 5 80 Out door Sept. 15 115

Example X General Procedure for Seed Soaking Treatment of AgriculturalCrops Using Watermelon Seeds

In this preferred embodiment of this invention, watermelon seeds weretreated with BMVE and planted alongside untreated seeds (control). Froman early seedling stage, the treated growth plants increased height aswell as foliage. The watermelon fruits of the treated were observablylarger than the control, the largest fruit form the treated plantweighed 16 pounds vs. 11 pounds for the largest control.

The results of the study of this embodiment showed that the treatedplants had increased overall biomass as compared to the control. Thestudy that encompasses this example is summarized in Table 10.

TABLE 10 Date of Date of Wt. of Melon Planting Treatment Dosage (ppm)Harvest (lbs) May 18 BMVE 5 Sept. 20 16 May 18 Water 0 Sept. 20 11

Example XI General Procedure for Seed Soaking Treatment of AgriculturalCrops Using Sunflower Seeds

In this preferred embodiment of the present invention, sunflower seedswere selected from the Melody variety and were soaked for three hours insolutions containing 0.05% Tween 20 and BMTA or BMPA, at a concentrationof 3 ppm. Another group of seeds of the same variety was soaked in asolution with only in 0.05% Tween 20 added (control). Following soaking,seeds were desiccated to storage moisture contents and BMTA, BMPA andcontrol seeds were sown in three twenty-five foot plots (Reps) each onMay 21 (plots were arranged in a randomized block design). On September16, sunflower heads were harvested, with a harvest of around 20 headsper plot/Rep on average. The heads were hand-threshed to collect seeds.Moisture, count, and weight data was collected on seeds from each head.

The results of the study of this embodiment showed that the treatedplants generally had increased moisture, seed count, and overall plantyield. The results from this example are encompassed in Table 11-A, andTable 11-B. Table 11-A shows mean seed moisture contents (% moisture),fresh seed weights, seed counts, seed weights at 6% moisture content(Wt. at 6% Moist; all data normalized to 6% seed moisture content toobtain yield data), and yield estimates in pounds based on planting rateof 20,000 seeds/acre for each Rep. Table 11-B shows the mean of the datapresented in Table 11-A over the three Reps for each treatment.

TABLE 11-A Yield @ Fresh Wt. 20,000 Treat- % Seed Wt. Seed at 6Plants/Acre ment Rep Moist (gm) Count % Moist (lbs) Control 1 10.5664.75 1081 61.54 2711 2 12.00 68.98 1065 64.01 2820 3 15.04 62.68 104156.68 2497 BMTA 1 13.10 77.35 1226 71.43 3147 2 13.01 73.58 1157 67.862989 3 14.82 61.64 942 55.94 2464 BMPA 1 13.52 78.24 1185 71.99 3171 213.93 65.01 977 59.42 2617 3 12.37 65.99 1052 61.46 2708

TABLE 11-B Fresh % Seed Wt Seed Wt at 6% Yield @ 20,000 Treatment Moist(g) Count Moist Plants/Acre (lbs) control 12.53 65.47 1062 60.74 2676BMTA 13.64 70.85 1108 65.08 2867 BMPA 13.27 69.75 1071 64.29 2832

Example XII General procedure for Seed Soaking Treatment of AgriculturalCrops Using Onion Seeds

In this preferred embodiment of the present invention, approximatelysixty onion seeds were soaked for three hours in water or solutioncontaining the KamTec bio-regulator BMCPA. Following soaking, five seedsfrom each seed soak treatment were sown in ten 5-inch pots (5 seeds eachin 10 total pots per seed soak treatment) containing Metro-Mix 200. Potscontaining seeds and Metro-Mix 200 media were randomized within seedsoak treatment (10 reps in a randomized block design) on a greenhousebench and watered daily to saturation in a growth regime set at 25° C.,14-hour days and 15° C., 10-hour nights. On Mondays and Fridays potswere fertilized with Peter's 20-10-20 at a rate of 250 ppm.

On days plants weren't fertilized, pots were saturated with wateradjusted to pH 6.8. Following about three weeks, pots were thinned toone plant per pot. Onion plants were harvested 47 days after planting.Digital images show each of the ten onion plants derived from the water(H₂O—left panel) and bio-regulator (right panel) seed soak treatments.

The results from a study of this embodiment generally showed increaseplant and root biomass. The results also showed increased yields ascompared to control groups. These results are visible in Table 12.

TABLE 12 Table 12, Mean root and shoot dry weights in grams on five N60Gand N73N corn plants 27 days after planting A 1 B 1 A 3 B 3 A 6 B 6Control ppm ppm ppm ppm ppm ppm N60G Roots 0.550 0.487 0.786 0.689 0.5930.631 0.705 Shoots 2.064 1.479 2.312 2.447 1.720 2.065 2.214 Root/ 0.2680.331 0.357 0.301 0.349 0.312 0.369 Shoot N73N Roots 0.803 1.100 0.8540.921 0.897 1.302 0.879 Shoots 3.583 4.185 3.391 2.936 3.426 4.979 3.186Root/ 0.227 0.284 0.260 0.315 0.268 0.282 0.286 Shoot

Example XIII General procedure for Seed Soaking Treatment ofAgricultural Crops Using Tomato Seeds

In a preferred embodiment of the present invention, tomato seeds aretreated with 3 ppm BMVE, and soaked for three hours before planting. Ina study of this embodiment, tomato seeds were treated and germinated inthe Cameron Highlands of Malaysia. Two months after planting, the plantswere observed and compared to a control group.

The results of the study of this embodiment showed that the treatedplants had increased roots, sprouts, and overall biomass as compared tothe control. The study that encompasses this example is summarized inTable 13.

TABLE 13 Date Soak time Date planted Observed Treatment Dosage (ppm)(hrs) May 23 Jul 23 BMVE 3 3

Example XIV General procedure for Seed Treatment of Agricultural CropsUsing Wheat Seed

In this preferred embodiment of the present invention, bio-regulatorsolutions can be used with fungicides and insecticides. Thebio-regulator solution can be tank-mixed with most seed protectionproducts provided such products are miscible in water and labeled forslurry application directly on seed. Compatibility can be checked bymixing a small amount of each product together to confirm suitability ofslurry application prior to application.

In another embodiment, the bio-regulator solutions can also be used withbiological products. They can be sequentially or simultaneously appliedwith most biological products when mixed in separate mix tanks. Directtank-mixing or mixing the bio-regulator solution in the same tank withbiological products such as legume inoculants, beneficial fungus, andother live microorganisms is not recommended as the bio-regulatorsolution might reduce the viability of the micro-organisms with directlymixed slurries. Preferred amounts used in these embodiments are threeounces per 100 pounds of seed.

Studies of these embodiments were carried out in the followinglocations: Imperial, Nebr.; Colby, Kans.; Manter, Kans.; Newton, Kans.;Osborne, Kans.; Palmer, Kans.; Sedgwick, Kans.; and, Enid, Okla. At eachof these locations soil analysis was performed and variouscharacteristics including, but not limited to, pH and mineral content,were recorded. These studies also included seed from the followingvarieties: WB-Armour, AP-Art, WB-Cedar, WB-Santa Fe, KSU FoundationEverest, WB-Hitch (later maturity), and WB-Winter Hawk (later maturity).

The results of the study of this embodiment showed that the treatedplants had increased overall yield and biomass as compared to thecontrol. The study that encompasses this example is summarized in FIG.8. FIG. 8 includes yields from each of the locations in units ofbushels.

2. General Procedure for Seed Soaking Treatment of for Growth Inhibitionof Agricultural Crops and Flora Seeds:

In a preferred embodiment of the present invention, amounts orconcentrations of the Ester Compounds, the BMIA Compounds, or the SaltCompounds are added to bio-regulatory solution in excess of thoseamounts for growth enhancement. In another embodiment, amounts orconcentrations of the Ester Compounds, the BMIA Compounds, or the SaltCompounds are added to bio-regulatory solution to encourage adetrimental gene expression in a particular environment during burndown.In another embodiment, said compositions are combined with herbicides inorder to enhance the effects of the herbicides during burndown.

Example XV Objective

In this preferred embodiment of the present invention, non-Roundup ReadyNE3001 soybean plants were treated with BMVE and glyphosate for thepurpose of determining whether BMVE could enhance the effects ofglyphosate on glyphosate-susceptible plants. These observations will beuseful in burndown scenarios.

Methods

Non-Roundup Ready soybean variety, NE3001 (NE3001 seeds were supplied byDr. Tom Clemente of the UNL Plant Transformation Core ResearchFacility). Seeds were sown into 224 5 inch pots (224 total pots with 4seeds each) containing soybean potting mix (37.5% Canadian Peat, 37.5%Coarse Vermiculite, 15% Masonry Sand, 5% Screened Topsoil, 3.63%Waukesha fine lime, 0.46% Micromax, 0.68% AquaGro, and 0.23% Hi YieldIron Plus). Pots were placed on benches inside a UNL Beadle Centergreenhouse bay set at 25° C., 14-hour days and 15° C., 10-hour nights.Pots were watered on Monday, Wednesday, and Friday. The irrigationschedule involved alternating water, 500 ppm Ca, and 350 ppm N and 60ppm Fe.

Two hundred and twenty-four pots were sown. At final stand, low seedlingemergence/normal seedling percentages were apparent over the 224 pots.Empty pots and pots containing abnormal seedlings were discarded. Potscontaining multiple normal seedlings were used for transplantingpurposes. Following transplanting, pots containing normal seedlings atsimilar stages of development were thinned to one plant per pot.Following the selection and transplanting process only 76 potscontaining normal seedlings remained. Of the 76 pots, 64 were utilizedin a spray table test.

NE3001 soybean plants at the sixth trifoliate leaf stage were sprayed ona spray table set to apply either 0.05% Tween 20, 5 ppm BMVE and 0.05%Tween 20, 50 ppm BMVE and 0.05% Tween 20, 100 ppm BMVE and 0.05% Tween20, 5 ppm BMVE and 0.5 quart of Nufarm Credit® glyphosate formulation,50 ppm BMVE and 0.5 quart of Nufarm Credit® glyphosate formulation, 100ppm BMVE and 0.5 quart of Nufarm Credit® glyphosate formulation, 5 ppmBMVE and 0.75 quart of Nufarm Credit® glyphosate formulation, 50 ppmBMVE and 0.75 quart of Nufarm Credit® glyphosate formulation, 100 ppmBMVE and 0.75 quart of Nufarm Credit® glyphosate formulation, 5 ppm BMVEand 1 quart of Nufarm Credit® glyphosate formulation, 50 ppm BMVE and 1quart of Nufarm Credit® glyphosate formulation, or 100 ppm BMVE and 1quart of Nufarm Credit® glyphosate formulation in 5 gallons of water peracre. Eight days after spray application the treated plants were given arating on a 1 to 5 scale; 1=normal soybean plant development/color,2=visual stunting of growth and <10% of the leaves exhibiting chlorosis,3=10 to 50% of the leaves exhibiting chlorosis, 4=>50% of the leavesexhibiting chlorosis, and 5=>50% of the leaves exhibiting chlorosis andsenescence. The spray formulations and ratings are listed in Table 15-A.Response of non-Roundup Ready soybeans to bio-regulator formulationseight days following spray application is shown in Table 15-B. NE3001soybean plants were grown to the sixth trifoliate leaf stage in agreenhouse regime set at 25° C., 14-hour days and 15° C., 10-hour nightsand then either not sprayed (control) or sprayed on a spray table set toapply 5, 50, or 100 ppm of BMVE in 5 gallons of water per acre.Application of the bio-regulator BMVE was shown to enhance the activityof glyphosate.

In some embodiments, particular bio-regulators may be particularlyeffective for enhancing herbicidal activity. For example, in someexperiments, at least one of CPTA, COPTA, DIPTA and DCPTA were effectivefor enhancing herbicidal activity.

TABLE 15-A Spray Formulation Rating 0.05% Tween 20 1 5 ppm BMVE and0.05% Tween 20 1 50 ppm BMVE and 0.05% Tween 20 1 100 ppm BMVE and 0.05%Tween 20 1 0.5 quart of Nufarm Credit ® 2 0.75 quart of Nufarm Credit ®2 1 quart of Nufarm Credit ® 3 5 ppm BMVE and 0.5 quart of NufarmCredit ® 2 50 ppm BMVE and 0.5 quart of Nufarm Credit ® 2 100 ppm BMVEand 0.5 quart of Nufarm Credit ® 2 5 ppm BMVE and 0.75 quart of NufarmCredit ® 3 50 ppm BMVE and 0.75 quart of Nufarm Credit ® 4 100 ppm BMVEand 0.75 quart of Nufarm Credit ® 3 5 ppm BMVE and 1 quart of NufarmCredit ® 5 50 ppm BMVE and 1 quart of Nufarm Credit ® 5 100 ppm BMVE and1 quart of Nufarm Credit ® 5

TABLE 15-B Fresh Pod Dry Pod Height Node Pod #/ Weight/ Weight/ (cm) #plant Plant (g) Plant (g) Control Mean 60.7 13.8 98.0 63.5 11.9 Std dev3.8 0.5 9.6 12.0 2.9 5 ppm B Mean 61.8 14.0 80.0 56.4 10.5 Std dev 7.71.2 12.1 22.9 4.6 50 ppm B Mean 64.3 13.8 91.0 55.9 9.5 Std dev 6.4 0.59.4 12.1 2.4 100 ppm B Mean 62.7 13.5 96.3 49.9 8.7 Std dev 7.4 1.3 9.07.4 2.2

Example XVI

In another preferred embodiment of the present invention, the effect oftreating Marestail weeds and Velvet Leaf weeds with a bio-regulatoraccording to one embodiment of the present invention were studied. Inthe study of the Marestail weeds there was a control group that wastreated with spray, containing 41% glyphosate, at levels of 1.25qt/acre. The test group was treated with spray at levels of two gallonsper acre, the spray containing 50 ppm of a bio-regulator+1 qt/acreglyphosate. Observations were made at 10 and 16 days after sprayapplication. Marestail weeds were treated twice. After treatments, itwas generally observed that the treated group appeared significantlymore stressed, had decreased vigor and pigmentation, and reducedbiomass.

In the study of the Velvet Leaf weeds, a process similar to the processfor Marestail weeds was followed. Velvet Leaf weed test groups weretreated with the same formulations and amounts of glyphosate andbio-regulator sprays, and control groups were treated with the sameformulation and amount of glyphosate. Again, after treatments, it wasgenerally observed that the treated group appeared significantly morestressed, had decreased vigor and pigmentation, and reduced biomass.

In at least one embodiment, bio-regulators according to embodiments ofthe present invention may increase the effectiveness of herbicides knownin the art. A person skilled in the art may appreciate that usebio-regulation to increase herbicide phytotoxicity may reduce theoverall need for herbicide, and thereby reduce the environmental andeconomic impact of such herbicides.

It is believed that the present invention and many of its attendantadvantages will be understood by the foregoing description ofembodiments of the present invention, and it will be apparent thatvarious changes may be made in the form, construction, and arrangementof the components thereof without departing from the scope and spirit ofthe invention or without sacrificing all of its material advantages. Theform herein before described being merely an explanatory embodimentthereof, it is the intention of the following claims to encompass andinclude such changes.

What is claimed is:
 1. A composition to be applied to plants and plantseeds comprising: an ester of the form

a wetting agent; an adjuvant; and a herbicide, wherein: R₁ is a loweralkyl group containing between one and six carbon atoms, and a benzylsubstituent; R₂ is a lower alkyl group containing between one and sixcarbon atoms, and a benzyl substituent; R₃ is one of a lower alkylgroup, cyclopropyl, phenyl and alkyl substituted phenyl; n is an integervalue from zero to four; the ester comprises at least one of2-(N-Methylbenzylaminoethyl)-2-methylpropanoate (BMBE),2-(N-Methylbenzylaminoethyl)-3-methylbutanoate (BMVE),2-(N-Methylbenzylaminoethyl)-2,2-dimethylpropanoate (BMPE),2-(N-Methylbenzylaminoethyl)-3,3-dimethylbutanoate (BMTE),2-(N-Methylbenzylaminoethyl)phenylacetate (BMBA),2-(N-Methylbenzylaminoethyl)phenoxyacetate (BMPA), and2-(N-Methylbenzylaminoethyl) cylcopropanoate (BMCPA); the adjuvantenhances uptake of the ester, sufficient to modify at least one geneexpression in a plant; and the composition produces a phytotoxic effectin at least one plant.
 2. The composition of claim 1, wherein theadjuvant is at least one of B-cyclodextrin and polyoxyethlene (20)sorbitan monooleate.
 3. The composition of claim 1, further comprisingat least one of polyoxythylene glycol p-isooctylphenylether,chlorothalonil and a mixture of polyalkyleneoxide modifiedpolydimethylsiloxane and polyoxypropylene block polymers.
 4. Thecomposition of claim 1, wherein the composition is formulated to modifyat least one gene expression in a plant.
 5. The composition of claim 1,wherein the composition is formulated to penetrate a seed.
 6. Acomposition to be applied to plants and plant seeds comprising: anaqueous solution comprising an ester of the form

wherein: R₁ is a lower alkyl group containing between one and six carbonatoms, and a benzyl substituent; R₂ is a lower alkyl group containingbetween one and six carbon atoms, and a benzyl substituent; R₃ is one ofa lower alkyl group, cyclopropyl, phenyl and alkyl substituted phenyl;the ester comprises at least one of2-(N-Methylbenzylaminoethyl)-2-methylpropanoate (BMBE),2-(N-Methylbenzylaminoethyl)-3-methylbutanoate (BMVE),2-(N-Methylbenzylaminoethyl)-2,2-dimethylpropanoate (BMPE),2-(N-Methylbenzylaminoethyl)-3,3-dimethylbutanoate (BMTE),2-(N-Methylbenzylaminoethyl)phenylacetate (BMBA),2-(N-Methylbenzylaminoethyl)phenoxyacetate (BMPA), and2-(N-Methylbenzylaminoethyl) cylcopropanoate(BMCPA); n is an integervalue from zero to four; and the composition produces a phytotoxiceffect on a class of plants.
 7. The composition of claim 6, furthercomprising an adjuvant, wherein the adjuvant is at least one ofB-cyclodextrin and polyoxyethlene (20) sorbitan monooleate.
 8. Thecomposition of claim 6, further comprising at least one ofpolyoxythylene glycol p-isooctylphenylether, chlorothalonil and amixture of polyalkyleneoxide modified polydimethylsiloxane andpolyoxypropylene block polymers.
 9. The composition of claim 6, whereinthe composition is formulated to modify at least one gene expression ina plant.
 10. A composition to be applied to plants and plant seedscomprising: an ester of the form

wherein: R₁ is one of a lower alkyl group containing between one and sixcarbon atoms, and a benzyl substituent; R₂ is one of a lower alkyl groupcontaining between one and six carbon atoms, and a benzyl substituent;R₃ is one of a lower alkyl group, cyclopropyl, phenyl and alkylsubstituted phenyl; the ester comprises at least2-(N-Methylbenzylaminoethyl)-3-methylbutanoate (BMVE); and n is aninteger value from zero to four.
 11. The composition of claim 10,wherein the composition comprises2-(N-Methylbenzylaminoethyl)-2-methylpropanoate (BMBE).
 12. Thecomposition of claim 10, wherein the composition comprises2-(N-Methylbenzylaminoethyl)-2,2-dimethylpropanoate(BMPE).
 13. Thecomposition of claim 10, wherein the composition comprises2-(N-Methylbenzylarninoethyl)-3,3-dimethylbutanoate (BMTE).
 14. Thecomposition of claim 10, wherein the composition comprises2-(N-Methylbenzylaminoethyl)-4-methylbenzoate (BMTA).
 15. Thecomposition of claim 10, wherein the composition comprises2-(N-Methylbenzylarninoethyl)phenylacetate (BMBA).
 16. The compositionof claim 10, wherein the composition comprises2-(N-Methylbenzylaminoethyl)phenoxyacetate (BMPA).
 17. The compositionof claim 10, wherein the composition comprises2-(N-Methylbenzylaminoethyl)cylcopropanoate (BMCPA).
 18. The compositionof claim 10, wherein the composition comprises2-(N-Methylbenzylaminoethyl)-4-bromobenzoate (BMPBA).
 19. Thecomposition of claim 10, wherein the composition is formulated toproduce a phytotoxic effect in the plant.